Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242;Department of Immunology, Third Military Medical University, Chongqing 400038, People’s Republic of China; and

Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242;Interdisciplinary Immunology Graduate Program, Carver College of Medicine, University of Iowa, Iowa City, IA 52242

Abstract

T cell factor (TCF)-1 and lymphoid enhancer-binding factor (LEF)-1 transcription factors have redundant roles in promoting thymocyte maturation. TCF-1 has been recently shown to critically regulate memory CD8+ T cell differentiation and persistence. The complete spectra of regulatory roles for TCF-1 and LEF-1 in CD8+ T cell responses are yet unknown. We conditionally targeted LEF-1, and by combination with germline deletion of TCF-1, we found that loss of both factors completely abrogated the generation of KLR G1loIL-7Rα+ memory precursors in effector CD8+ T cell populations in response to Listeria monocytogenes infection. Whereas CD8+ effectors deficient for TCF-1 and LEF-1 retained the capacity to express IFN-γ, granzyme B, and perforin, they were defective in TNF-α production. In the memory phase, the Ag-specific CD8+ T cells lacking TCF-1 and LEF-1 exhibited an effector phenotype and were severely impaired in secondary expansion upon rechallenge. Thus, TCF-1 and LEF-1 cooperatively regulate generation of memory precursors and protective memory CD8+ T cells.

Introduction

Cytotoxic CD8+ T cells are essential for controlling intracellular pathogens, including viruses and some bacteria. Upon encountering their cognate Ag, naive CD8+ T cells are activated and expanded into clonal effector T cells. A small fraction of CD8+ effectors then gives rise to memory T cells that confer enhanced protections against the same pathogen (1, 2). Both effector and memory CD8+ T cells are heterogeneous. Effector CD8+ T cells with increased expression of KLRG1 and decreased expression of IL-7Rα are considered to be short-lived and terminally differentiated; in contrast, KLRG1loIL-7Rα+ effectors demonstrate increased potential of generating long-lasting memory CD8+ T cells and are therefore proposed to be memory precursors (3, 4). Among memory CD8+ T cells, CD62L+CCR7+ central memory T cells exhibit a greater capacity of homeostatic proliferation, whereas CD62L−CCR7− effector memory T cells decay over time (5).

Transition of naive into effector and memory CD8+ T cells is accompanied by diversification of the T cell transcriptome, and several transcription factors are known to direct effector and memory T cell differentiation. T-bet, Blimp-1, and Id2 are highly induced upon T cell activation, and their expression is more enriched in the terminally differentiated CD8+ effectors than in the KLRG1loIL-7Rα+ memory precursors (3, 6–8). In contrast, the memory precursors express higher levels of Bcl-6, T cell factor (TCF)-1, and Id3 than do the terminally differentiated effectors (8).

TCF-1 and lymphoid enhancer-binding factor (LEF)-1, encoded respectively by Tcf7 and Lef1, are transcription factors that act downstream of the canonical Wnt signaling pathway (9, 10). These two factors have known redundant roles in T cell development (11, 12). We and others have recently demonstrated that TCF-1 deficiency compromised memory CD8+ T cell differentiation and persistence (13, 14). To address possible compensatory roles between TCF-1 and LEF-1, we conditionally targeted the Lef1 locus. By combination with germline deletion of Tcf7, in this study we demonstrate that TCF-1 and LEF-1 are essential for generation of memory precursors and functional memory CD8+ T cells.

Materials and Methods

Mice

Tcf7−/− and Gzmb-Cre transgenic mice were from Hans Clevers and Joshy Jacob, respectively (15, 16). Rosa26-enhanced GFP (EGFP) reporter mice were from The Jackson Laboratory. The Lef1-floxed strain was generated in-house (17). All of the animals were housed and handled following protocols approved by the Institutional Animal Care and Use Committee at the University of Iowa.

Infections

Mice were initially i.v. infected with ∼0.5 LD50actA–Listeria monocytogenes-expressing OVA (LM-OVA) (18). For recall response, the immune mice were i.v. infected with 10 LD50 virulent LM-OVA, and CFUs in the spleen and liver were determined (19).

Flow cytometry and cell sorting

Cell surface staining, intracellular staining for cytokines, intranuclear staining for eomesodermin (Eomes) and T-bet, and OVA257–264 (SIINFEKL)-MHC class I tetramer staining were performed as previously described (14, 19). The flow data were analyzed using FlowJo software (Tree Star). Tetramer-stained CD8+ effector or memory T cells from the spleens were sorted on a FACSAria.

Chromatin immunoprecipitation and quantitative RT-PCR

A LEF-1 Ab (C18A7; Cell Signaling Technology) was used in chromatin immunoprecipitation on CD8+ T cells as described (14). RNA extraction, reverse transcription, and quantitative PCR were performed as described (14).

Deficiency in TCF-1 and/or LEF-1 impaired expansion and function of effector CD8+ T cells and generation of memory precursors. (A) Kinetics of CD8+ T cell responses in PBLs. Mice were infected with actA– LM-OVA, and OVA-specific CD8+ T cells in the PBLs were detected by peptide-stimulated IFN-γ production. The frequency of IFN-γ+GFP+ cells among CD8+ T cells was shown. Data are representative of three independent experiments (n ≥ 4 for each time point). (B) Numbers of OVA-specific CD8+ effectors in the spleen, as determined on day 7 postinfection. (C) Characterization of CD8+ effectors in the absence of TCF-1 and/or LEF-1. On day 7 postinfection, OVA-specific IFN-γ+GFP+CD8+ T cells were detected in the spleen and fractionated based on KLRG1 and IL-7Rα expression or detected for TNF-α and granzyme B induction. The gating is based on isotype control staining, and the frequency of each subset is shown in representative contour plots from three independent experiments (n ≥ 6). (D) Cumulative frequency of KLRG1loIL-7Rα+ memory precursors in IFN-γ+GFP+CD8+ effector T cells. (E) Perforin expression in CD8+ effectors. Tetramer+GFP+CD8+ effector T cells were sorted from the spleens on day 7 postinfection, and Prf1 expression was measured by quantitative RT-PCR. The primers are 5′-GATGTGAACCCTAGGCCAGA-3′ and 5′-GGTTTTTGTACCAGGCGAAA-3′. Data in (B), (D), and (E) are means ± SD from three to four independent experiments (n ≥ 6). **p < 0.01, ***p < 0.001 by Student t test.

To avoid perinatal lethality, we conditionally targeted the Lef1 gene by inserting 2 LoxP sites to flank exons 7 and 8, which encode the DNA-binding HMG domain of LEF-1 (17). To circumvent the impact of TCF-1 and LEF-1 double deficiency on T cell development and to specifically investigate their roles in mature T cells, we use a Cre transgene driven by human granzyme B promoter (Gzmb-Cre) (16). In this system, LEF-1 expression remains intact in mature CD8+ T cells, and excision of the floxed Lef1 allele (Lef1FL/+) occurs only after the naive T cells are activated (8). We also used a Rosa26-EGFP reporter strain in which GFP is only expressed after Cre-mediated excision of an intervening floxed STOP sequence (20). This reporter thus permanently marks Ag-experienced CD8+ T cells with effective excision of the floxed alleles. Upon proper crossing, we obtained Gzmb-Cre+Rosa26-EGFP+Lef1FL/FL and Gzmb-Cre+Rosa26-EGFP+Lef1FL/FLTcf7−/− mice (hereafter designated as Lef1−/− and double knockout [dKO] mice, respectively). For control and Tcf7−/− mice, we used those positive for Gzmb-Cre and Rosa-EGFP reporter genes for a consistent comparison. On day 7 postinfection with LM-OVA, 70–95% of the OVA-specific effector CD8+ T cells, detected either with OVA peptide-stimulated IFN-γ production or SIINFEKL-MHC class I tetramer, were positive for GFP in all genotypes examined (Supplemental Fig. 1A, 1B). We also sorted tetramer+GFP+CD8+ effectors and found that the Lef1 transcripts were decreased by ∼80% in Lef1−/− and dKO mice (Supplemental Fig. 1D). Assuming the remaining Lef1 transcripts were all derived from one undeleted “floxed” Lef1 allele in a single cell, ∼80% of Lef1−/− and dKO CD8+ effectors should have complete inactivation of LEF-1.

To determine the impact of TCF-1 and LEF-1 deficiency on CD8+ T cell responses, we tracked OVA-specific IFN-γ+GFP+CD8+ T cells in both PBLs and spleens. In the PBLs, whereas TCF-1 and/or LEF-1 deficiency affected the magnitude of CD8+ effector expansion to a different extent, the peak expansion in all genotypes occurred on day 7 postinfection (Fig. 1A). Consistent with our previous findings (14), TCF-1 deficiency reduced frequency of CD8+ effectors in the PBLs as well as their numbers in the spleen by >50% (Fig. 1A, 1B). Although ablation of LEF-1 only minimally affected the frequency of CD8+ effectors in the PBLs (Fig. 1A), it significantly reduced the number of effector CD8+ T cells in the spleen (Fig. 1B). These results suggest that LEF-1 is also required for optimal expansion of CD8+ effectors, and this is in contrast to the observation that LEF-1 is dispensable for normal T cell development (12). In contrast, deletion of both TCF-1 and LEF-1 substantially reduced the frequency of OVA-specific CD8+ effectors detected in PBLs (Fig. 1A). In the spleen, albeit there was a trend of further reduction of CD8+ effectors in dKO mice compared with Lef1−/− or Tcf7−/− mice, the additive effect of TCF-1 and LEF-1 double deficiency was not evident (Fig. 1B). We also used SIINFEKL-MHC class I tetramer to detect OVA-specific CD8+ effectors and confirmed those findings using IFN-γ–based functional measurements (Supplemental Fig. 1C). These data collectively suggest that TCF-1 and LEF-1 transcription factors are necessary for optimal expansion of effector CD8+ T cells, and that deletion of both factors did not completely abrogate CD8+ effector differentiation.

Loss of TCF-1 and LEF-1 abrogated generation of memory precursors

In addition to quantitative measurements, we next performed in-depth phenotypic and functional analysis of effector CD8+ T cells lacking TCF-1 and/or LEF-1. When fractionated based on KLRG1 and IL-7Rα expression, the KLRG1loIL-7Rα+ memory precursors were diminished by either LEF-1 or TCF-1 deficiency (Fig. 1C, 1D). Strikingly, the memory precursors were almost completely abrogated in dKO mice (Fig. 1C, 1D), indicating that the generation of memory precursors depends on TCF-1 and LEF-1. Additionally, although loss of either TCF-1 or LEF-1 alone exhibited little effect, the double deficiency substantially diminished production of TNF-α in CD8+ effectors (Fig. 1C). In contrast, TCF-1 and/or LEF-1 deficiency did not compromise the ability of CD8+ effectors in acquiring the cytolytic effector molecules such as granzyme B (Fig. 1C). In fact, the expression of perforin (encoded by Prf1) was most evidently increased in dKO CD8+ effectors (Fig. 1E).

After the peak response, a fraction of the CD8+ effector T cells survives the contraction and transition into memory phase. On day 35 postinfection, the numbers of OVA-specific memory CD8+ T cells were reduced in the Tcf7−/− spleens, as detected by OVA peptide-stimulated IFN-γ production or MHC class I tetramer (Fig. 2A, Supplemental Fig. 1E–G). Double deletion of TCF-1 and LEF-1 further reduced the numbers of memory CD8+ T cells, albeit the reduction did not reach statistical significance compared with TCF-1 single deficiency (Fig. 2A, Supplemental Fig. 1G). It is noteworthy that loss of both TCF-1 and LEF-1 abrogated generation of KLRG1loIL-7Rα+ memory precursors in the effector clonal expansion phase (Fig. 1C, 1D). Although the memory precursors are considered to have increased potential to give rise to memory CD8+ T cells, in the context of TCF-1 and LEF-1 double deficiency, the dKO OVA-specific CD8+ T cells detected at the early memory phase are most likely derived from the KLRG1+IL-7Rα– effector CD8+ T cells. In line with this notion, the dKO memory CD8+ T cells exhibited almost exclusively a KLRG1+ effector phenotype (Fig. 2B, 2C). Although not affected in granzyme B expression, the dKO memory CD8+ T cells manifested decreased production of TNF-α and failed to produce IL-2 (Fig. 2D). We also sorted tetramer+GFP+CD8+ memory T cells to assess Lef1 excision and found that the cells with complete deletion of Lef1 transcripts were reduced to ∼60% at the memory stage, compared with ∼80% excision in effectors (compare Supplemental Fig. 1H with Supplemental Fig. 1D). This suggests that the cells that escaped Lef1 excision may have had growth/survival advantage during effector-to-memory transition, and hence the defects of memory CD8+ T cells in Lef1−/− and dKO mice may have been underestimated in this experimental system.

We previously showed that Eomes is a direct TCF-1 target gene in memory CD8+ T cells (14). Eomes is upregulated in CD8+ effectors and retained at high levels in memory T cells (21). At the effector phase, Eomes was only minimally affected by loss of TCF-1 or LEF-1 alone, but it was evidently reduced in dKO CD8 effectors; in contrast, T-bet expression was not affected by loss of TCF-1 and/or LEF-1 (Supplemental Fig. 2A). Thus, deficiency in both TCF-1 and LEF-1 had an early impact on proper upregulation of Eomes in CD8+ effectors, impairing generation of functional memory CD8+ T cells. At the memory phase, TCF-1 deficiency reduced Eomes expression as expected, and interestingly, loss of LEF-1 alone also modestly reduced Eomes expression (Supplemental Fig. 2B). We previously reported direct binding of TCF-1 to four upstream regulatory regions in the Eomes gene (14). By chromatin immunoprecipitation on CD8+ T cells, we found that LEF-1 occupied the same regulatory sequences (Supplemental Fig. 2C). These data indicate that both TCF-1 and LEF-1 contribute to positive regulation of Eomes during CD8+ T cell response.

Memory T cells confer enhanced protection upon re-encountering the same Ag. When challenged with virulent LM-OVA, whereas naive mice showed uncontrolled bacteria growth, control immune mice completely cleared L. monocytogenes in the spleen and largely in the liver (Fig. 3A, 3B). In contrast, the bacteria were detected in the spleen of 50% of dKO mice, and in the liver of dKO mice, the bacteria burden was >1 order of magnitude higher than in the control mice (Fig. 3A, 3B). To further investigate the less efficient bacterial clearance in the absence of TCF-1 and LEF-1, we tracked the recall response of memory CD8+ T cells. In PBLs, the secondary expansion of dKO memory CD8+ T cells was greatly diminished, as measured by absolute frequency or relative expansion after normalizing to the starting point (Fig. 3C, 3D). When examined in the spleen, the OVA-specific dKO memory CD8+ T cells were greatly impaired in generating secondary effectors in terms of absolute counts as well as relative expansion (Fig. 3E, 3F). Collectively, most of these defects in dKO CD8+ memory were more severe than those observed in Tcf7−/− memory CD8+ T cells, indicating that TCF-1 and LEF-1 have redundant roles in regulating CD8+ responses, in addition to their well-known roles in cooperatively promoting thymocyte maturation (12).

Loss of TCF-1 and LEF-1 greatly impaired recall response by the primary memory CD8+ T cells. (A and B) Clearance of virulent LM-OVA by memory CD8+ T cells. Naive or immune mice (day 35 postinfection) were challenged with virulent LM-OVA, and 3 d later, CFUs were determined in the liver and spleen. Shown are cumulative data from two experiments. Data are reported as CFU numbers (means ± SD) per spleen (A) or per gram of liver (B) from each organ with positive detection of LM-OVA. Frequency of animals with positive detection of the bacteria is marked on top of the bar. LOD, limit of detection. (C and D) Secondary CD8+ T cell expansion in PBLs. OVA-specific CD8+ T cells were tracked in the PBLs after secondary challenge. (C) Frequency of IFN-γ+GFP+ cells among CD8+ T cells. (D) Relative expansion of secondary CD8+ effectors after normalization to starting memory CD8+ T cell frequency. Data are representative of three independent experiments (n ≥ 7). (E) Numbers of secondary effector CD8+ T cells in the spleens, as determined on day 5 after the rechallenge. Data are means ± SD from three independent experiments (n ≥ 5). *p < 0.05, **p < 0.01, ***p < 0.001. (F) Relative expansion of secondary CD8+ effectors in the spleen. The numbers of secondary CD8+ effectors [as in (E)] was normalized to those of primary memory CD8+ T cells (as in Fig. 2A) to calculate the relative expansion.

In summary, using the newly established LEF-1 conditional knockout mouse model, our study revealed unique requirements of TCF-1 and LEF-1 transcription factors in regulating mature CD8+ T cell responses. To our knowledge, this is the first demonstration that deletion of transcription factors (i.e., TCF-1 and its relative LEF-1) in activated T cells completely abrogates the generation of KLRG1loIL-7Rα+ memory precursors. This study further uncovers essential roles of TCF-1 and LEF-1 in TNF-α production and proper upregulation of Eomes in CD8+ effectors. Upon transitioning to the memory phase, the Ag-specific CD8+ T cells lacking both factors exhibit an effector phenotype and fail to effectively acquire functions characteristic of memory T cells, such as rapid secondary expansion and effective control of pathogens. These observations reveal that deficiency in TCF-1/LEF-1 causes the most profound defects in CD8+ T cell responses among all the transcription regulators studied in vivo thus far. Our data collectively indicate that TCF-1 and LEF-1 are necessary for optimal expansion of CD8+ effectors and are indispensable for generation of memory precursors as well as further maturation and acquisition of CD8+ memory functionality. These findings thus identify TCF-1 and LEF-1 as key regulatory nodes in enhancing T cell immunity against infectious agents and malignant cells.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Drs. John Harty and Vladimir Badovinac for providing LM-OVA and SIINFEKL tetramers and for critical reading of this manuscript. We thank Drs. Hans Clevers (Hubrecht Institute, Utrecht, The Netherlands) and Joshy Jacob (Emory University, Atlanta, GA) for providing the Tcf7−/− and Gzmb-Cre transgenic mice, respectively.

Footnotes

This work was supported by National Institutes of Health Grants HL095540 and AI080966 and by American Cancer Society Grant RSG-11-161-01-MPC.